Reagent and method for determining the coagulability of blood



April 20, 1965 P. A. OWREN 3,179,567

REAGENT AND METHOD FOR DETERMINING THE COAGULABILITY OF BLOOD Filed May 12, 1959 FIG]. 120:

5 1o 20 so 100% mysnTQR PA L ARA/0K OWREN United States Patent 3,179,567 REAGENT AND METHOD FOR DETERMINING THE COAGULABILITY OF BLOOD Paul Amer Owren, Blommenholm, Baerum, Norway Filed May12, 1959, Ser. No. 812,682

Claims priority, applicatigmoNorway, May 16, 1958,

12 ,1 4 14 Claims. (Cl. 167-845) Oral anticoagulants (dicumarol and dicumarol derivates, phenylindandione and similar substances) cause a reduction in the coagulability of the blood and are ex tensively used today in the treatment of thrombotic conditions. Prophylaxis against thrombosis in arteriosclerotic disease (coronary infarct, angina pectoris, cerebral and peripheral arterial thromboses) is being more and more generally adopted.

Efiective anticoagulant treatment requires regular determinations of the coagnlability of the blood. It is necessary to adjust the dosage .to the individual case so as to avoid, on the one hand, excessive amounts which may result in bleeding complications, and,"on the other, too small a dosage which will fail to be effective.

With previously described techniques for this control it has not been possible in each individual case to determine the coagulability of the blood with sufiicient certainty so that these dosage difficulties are solved; there therefore exists a great need for methods which make accurate control possible. b

By means of the reagent comprising the present invention, this problem is solved in a satisfactory way, as will appear from the following description of. the invention.

In order to make it easier to understand the invention an account will be given of the action of anticoagulant agents.

Coagulation of the blood is effected by means of two partly separate coagulation systems: the external and the internal system. The peroral anticoagulant agents cause a decrease in the concentration of the following four coagulation factors in the blood: prothrombin, Stuart-factor, pro-convertin and antihemophilia-B-factor. Of these factors, prothrombin and Stuart-factor are involved in both systems; proconvertin acts only in the external system; and antihemophilia-B-factor participates only in the internal system. This is shown in the following scheme:

Coagulation system of the blood The "External System The Internal System (Cephalin Time) (Thromboplastin time) Blood platelet factor 3 (Cephalin), Antihemophilia-A-factor (Ali. G.) Antihemopl1ilia-Bfactor(P.T.C.). Antihemophilia-Ciaetor (P.T.A.). Hageman-iactor.

Staurt-faetor.

Prothrombin. Proaceeleriu. Fibrinogeu.

Tissue-thromboplastin.

Proeonvertin.

amass? Patented Apr. 20, 1965 ice The accompanying FIGURE 1 shows. the sensitivity of these three methods to decreasing quantities of antihemophilia-B-factor. Concentration of antihemophilia-B- factor relative to the other factors isgiven as a percentage of the normal value along the abscissa. .The coagolation time in seconds (20 .to see.) is plotted on the ordinate.

The study was made by mixing hemophilia-B-plasrna and normal plasma in various proportions.

Curve A shows the sensitivity of the new method to diminishing quantities of antihemophilia-B-factor.

.Curve B shoWs the sensitivity of the P-P-method and curve C shows the sensitivity of Quicks method to diminishing quantities of antihernophilia-B-.tactor.

The explanation of the differences seen in the curves shown in the figure is that in the P-P-method and in Quicks method a very active tissue-thromboplastin is added to the blood or plasma to be examined. 4

In Quicks method an extract of rabbit brain is used and in the P-P-method an extract of human brain. These very active thromboplastins cause the coagulation to occur exclusively via the external coagulation system, since clotting in this system is so rapid that it is over long before the internal system can become fully activated. Thus he normal coagulation time of a plasma following addition of an active thromboplastin, as in'Quicks method, is only 12-l3 seconds, whereas the internal syfi im, even .after addition of optimal cephalin concentration, requires at least 50 seconds to produce the formation of thrombin which in turn coagulates fibrinogen. Addition of such active thromboplastins completely circumyents the inter.- nal system. With Quicks method the coagulation ,time (which is often called the prothrombin time" .ohjthe thromboplastin time) is consequently perfectly .normal even when there is complete absence of antihemophilia- B-factor (cf. FIG..1). i

It should be pointed out that methods have been worked out for determining the activity of theinternal coagulation system, using cephalin instead of thrornh oplastin. These methods can, depending on the details of the system, be used for determination .of the various antihemophilic factors. However, rr ethods of this type are never used for control of anticoagulant treatment .for .two principal reasons. First, such cephalin times are very sensitive to a number of incidental effects; secondly, they are entirely unaffected by changes in the external coagulation system, of which yariations in proconvertin concentration maybe most important. l

The present invention provides a method which permits the practitioner to determine simultaneously the coagulability of the blood in both the external and the internal system with a single test. By .rnea1 1s ofthe new reagent the external and the internal coagulation system are started simultaneously and in such manner that .they proceed at approximately the same rate, with the result that the two systems exert fairly equal influences .onthe coagulation time of the system.

o) Previous methods for control of anticoagulant treatment Two principal methods have, as mentioned, been used hitherto for control of anticoagulant treatment: (1) Quicks method; (2) The P-P-method.

(l) Quicks methd.[Quick, A. 1.: Biol. Chem. J. 109, lxxiii (1935).]

Quicks method was originally introduced as a method for the determination of the prothrombin of the blood; the factors proconvertin, Stuart-factor and proaccelerin were at that time unknown. In this method calcium and the thromboplastin concentrations are kept constant.

The method has undergone minor changes. The latest detailed description is the following: Quick, A. 1.: Hemorrhagic Diseases, Lea and Fibiger, Philadelphia, 1957, pp. 379-387.

Reagents:

(1) Thromboplastin suspension: Extract of acetonedried rabbit brain. 7

(2) Calcium chloride solution 0.01 M.

Procedure: 0.1 ml. oxalated plasma is mixed with 0.1 ml. thromboplastin solution, placed in water-oath at 37 C. and recalcified with 0.1 ml. of 0.01 M calcium chloride solution, and the coagulation time or the thromboplastin time (also called Quicks prothrombin time) is determined.

Commentary: The method is sensitive only to the external coagulation system (of. the scheme in column 1). The internal coagulation system does not, as stated earlier, have an opportunity to influence the coagulation time.

Thromboplastin and calcium are the only factors which are kept constant in Quicks method. A change in any of the other factors of the external system: prothrombin, proconvertin, Stuart-factor, proaccelerin or fibrinogen, would also, however, be able to afieet the registered Quicks thromboplastin time. During anticoagulant treatment it is probable that proaccelerin and fibrinogen are relative- 1y constant; hence any prolongation of Quicks thromboplastin time will be due to a reduction of proconvertin, Stuart-factor, and/ or prothrombin. On the other hand the method is entirely insensitive to a reduction of antihemophilia-B-Factor. In hemophilia-B Quicks thromboplastin time? is normal (see FIG. 1). Therefore, if this method is used for control of the dosage in anticoagulant treatment, there is a risk that hemorrhage will occur because of a particularly large reduction of antihemophilia-B- factor, since such a change would not be revealed with this method. By specific quantitative determination of the various factors involved we have found that antihemophilia-B-factor is often reduced more markedly than the other factors, as can be seen from FIGURE 2. These results. are for phenylindanedione, but Dicumarol and Di cumarol derivates have essentially the same effect.

On the abscissa the numbers 830 indicate the dates in March 1957 when the tests were performed. The tests were concluded on April 2, 1957.

On the ordinate are given percentages (these are not absolute, 100% signifying normal concentration) of prothrombin, proconvertin and antihemophilia-B-factor found in the blood on the dates shown by analyses conducted during this investigation.

Curve D shows the percentage of prothrombin found in the blood; curve E, the percentage of proconvertin; and curve F, the percentage of antihemophilia-B-factor.

As can be seen, the level of the three mentioned factors was approximately 100% at the beginning of the tests. On March 9, 1957, the anticoagulant agent phenylindandione was begun in daily doses, the quantity of which can be seen in the area at the top of the figure. On the left of this area the vertical line represents 100 mg. phenylindandione. The quantity of each days dose appears at the vertical line drawn from the date in question on the abscissa. On March the administration of the anticoagulant agent was stopped and the percentage content of the factors then rose quickly.

The fact that Quicks method is not sensitive to reduc tion of antihemophilia-B-factor is thought to be one of the causes of the relatively large frequency of hemorrhagic complications when Quicks method is used for control of anticoagulant treatment.

Among other defects of this method may be mentioned the facts that it is insensitive to variations of all three of the factors in the range 50 to of normal, that the calcium concentration employed is not optimal if the hematocrit value of the blood is abnormal, and that the thromboplastin time is affected by the proaccelerin concentration in the plasma examined. Proaccelerin is unstable and is inactivated during storage of oxalated plasma; for this reason the method requires a relatively fresh blood sample.

(2) The P-P-method (prothrombin-proconvertin method) .-Owren, P.A.: The Coagulation of Blood, Acta Med. Scand. 128 (suppl. 194), p. 327 (1947); Owren, P.A.: Scand. J. Clin. Lab. Invest. 1, 81-83 (1949); Owren, PA;

and Aas, K.: Scand. J. Clin. Lab. Invest. 3, 201-208 Reagents:

(1) Thromboplastin suspension: Aqueous extract of human brain.

(2) Adsorbed bovine plasma.

(3) Calcium chloride solution of optimal concentration (normally 0.025 M).

Procedure: 0.2 ml. adsorbed bovine plasma is mixed with 0.2 ml. thromboplastin suspension and 0.2 ml. of a 1:10-dilution of the plasma to be investigated; the mixture is placed in water bath at 37 C. The coagulation time (P-P time) is recorded after recalcification with 0.2 ml. calcium chloride solution.

Commentary: As in the case of Quicks method, the P-P-method is a determination of the thromboplastin time, i.e. it is primarily a method for estimating the activity of the external coagulation system. The chief differences between the P-P-method and Quicks method are the following:

(1) A new reagent, adsorbed bovine plasma, is introduced in order to stabilize the concentration of proaccelerin and fibrinogen. The measurement is therefore independent of the concentration of these factors in the test plasma. Since the factors which are reduced during anticoagulant treatment are relatively stable during storage of blood and plasma, the method may be used on stored samples. 7

(2) A 10% dilution of the test plasma is used. This increases the sensitivity of the method, especially in the range 50-100%. Dilution of the test plasma also has the advantage that the risk of inoptimal calcium concentration with varying hematocrit values is far less than in Quicks method. The diluting also eliminates the effect of small amounts of heparin in the plasma to be examined.

As in the case of Quicks method, the PAP-method is sensitive to reductions of prothrombin, Stuartfactor and proconvertin. [Gonyea, L.M., l-ljort, P., Owren, P.A.: The Journal of Laboratory & Clinical Medicine, 48, 624- 633 (1956).]

The thromboplastin used in the P-P-'nethod (aqueous extract of human brain) exerts, in addition to its thromboplastin effect, a cephalin effect on the internal coagulation system. This effect is, however, not optimal. As a result of this cephalin effect, and of the dilution of the test plasma, it can be shown that the P-P-method possesses a certain, though exceedingly small, sensitivity to excessive reductions of antihemophilia-B-factor, if the external coagulation system is greatly impaired following anticoagulant treatment (see FIG. 1). [Waaler, B. A.: Scand. J. Clin. Lab. Invest. 9, 322-330 (1957).] From the point of view of clinical practice this sensitivity of the P-P-method to reductions of antihemophilia-B-factor is far too small to permit it to provide, certain protection against hemorrhagic complications due to severe depression of antihemophilia-B-factor.

From time to time other modif cations of Quicks method have been suggested, such, for instance, as employment of other thromboplastin preparations-drum rabbit lungs and human brain, some in acetone-dried form and others in frozen-dried form. Dilutions of the plasma to be investigated have also been used in order to increase the sensitivity of the method, especially in the range 50100% Variation of the calcium concentration relative to the hematocrit value of the blood have also been suggested in order to secure optimal recalcification. However, none of these modifications has altered the principle of the method, and none has involved any change in the methods defective sensitivity to reduction of antihemophilia-B-factor.

The reagents for the PP-methodthe adsorbed bovine plasma and the thromboplastin suspension-have been produced in frozen-dried form. However, this change has of course not altered the defective sensitivity of the method to reduction of antihemophilia-B-factor.

Method for control of the coagulability of the blood during anticoagulant treatment From the foregoing it will be seen that none of the hitherto available aids has been satisfactory for the determination of the coagulation activity of the blood in order to control anticoagulant treatment.

The following account describes the invention: a preparation which permits satisfactory determination of blood coagulability as a control of this treatment.

This procedure is based on an entirely new principle, namely: that a simultaneous and combined determination is made of the activity in the internal and in the external coagulation system. Hitherto we have not had any methed for such combined determination; the two coagulation systems have always been investigated separately.

The method is based on a reagent which initiates both coagulation systems simultaneously and in such a way that they both require approximately the same amount of time. To achieve this situation the internal coagulation system has been given optimal conditions for maximum speed by inclusion of cephalin from human brains or any other cephalin preparation which has the same activity, in the reagent in optimal concentration. The internal coagulation system alone will produce complete coagulation under these conditions in about 50 seconds.

At the same time the reagent contains a thromboplastin which starts the external coagulation system, but this thromboplastin is of such a nature that its maximal activity is low as compared with human brain thromboplastin and rabbit brain thromboplastin. As a result, the speed of the external coagulation system is considerably reduced as compared to the normal speed obtained with active thromboplastin preparations of the type used for Quiclrs method and the P-P-method. The thromboplastin used in this method should give a thromboplastin time with normal human plasma of 3550 seconds, as tested by Quicks method.

Theoretically a weak thromboplastin could be produced by dilution of an active thromboplastin (of the type used for Quiclrs method and the P-Pmethod), but such a procedure results in a labile and practically useless reagent because small changes in its activity give considerable oscillations in the registered times. The reagent therefore employs a thromboplastin which has a stable and low activity in optimal concentration and which has a relatively wide range of optimal concentrations. 7

As starting material for production of such a thrombo- Jplastin, lung or brain of certain species of animals (bovine, horse, swine, sheep, dog) are used. The thromboplastin produced is species specific and reacts only slowly with human proconvertin, as revealed by the thromboplastin time of 35-50 seconds as compared with the thromboplastin time of 13-15 seconds of human brain thromboplastin.

6 The combination in one reagent of a thromboplastin having such weak activity toward human plasma and of cephalin, both in the concentrations of. maximal activity,

has been found to result in the internal and the external coagulation systems having approximately equal influence on the coagulation time. This method is therefore sensitive to reductions both of antihernophilia-B-factor in the internal system and of proconvertin in the ex ternal system, in addition to Stuart-factor and prothronibin, which participate in both systems.

PEG. 1 shows the marked sensitivity of this method to large reductions of antihemophilia-B-fac:tor, in contrast to the very defective sensitivity of Quicks method and the very slight sensitivity of the P-P-method.

It follows from this that the principal drawbacks of the methods and reagents hitherto employed (Quicks method, the P-P-method) have in all essential respects been eliminated.

The invention is, further, based on the concept that the coagulation times determined should be independent of changes in all the coagulation factors which are not affected by antocoagulant treatment. This is accomplished by the inclusion in the reagent of all these factors in high concentrations (actual concentrations specified below). For this purpose a specially adsorbed plasma (bovine, horse, swine, sheep, dog) prepared similar to that as for the P-l method, is used. This adsorbed animal plasma contains high concentrations (minimal 90%relative to human plasma) of .antihernophilia- A-factor, antihemophilia-C-factor and Hageman-factor (of the internal system), and of proaccelerin and fabrinogen (which participate in both systems). It is, further, prepared in such a way that it does not contain perceptible amounts (as determined by the methods specifled below) of proconvertin, antihemophilia-B-factor, Stuart-factor or prothrombin-precisely the four factors which are reduced during anticoagulant treatment.

As a result, the reagent is specific for and sensitive to all the four factors which are reduced during anticoagulant treatment, and is at the same time uninfiuenced by variations in the other coagulation factors. The coagulation times which are registered by this method therefore provide an adequate index of the total effect which the anticoagulant agents have on the coagulation of the blood. Thedecisive difference from earlier methods is, as stated, the sensitivity of the new method to reduction of the concentration of antihemophilia- -factor.

Composition of the reagent The new reagent thus contains the following four components:

(l) Thromboplostin.-Every type of :thromboplastin may he used, provided it fulfills the following requirements: (4) The thromboplastin suspension is stable on storage at to C. for at least 24 hours. (b) The optimal dilution of the thromboplastin suspension, which produces the maximal activity for the preparation as tested on normal human plasma, gives a thromboplastin time between 39 and seconds as tested by the method of Quick. This ,thromboplastin time provides the desired balance between the internal and the external clotting systems.

Thromboplastin preparations having the specified activityhave beenprepared as follows:

(1) Extracts from bovine, horse, dog, swine and sheep brain. [Method0wren, P. A.: Scand. J. Clin. Lab Invest. 1, 81 0949).]

(2) Extracts from bovine, horse, swine, dog and sheep lungs. [MethodWare, A. G. and Seegers, W. 1-1.: Am. J. Clin. Path, 19,471 (1949).]

Examoles of thromboplastin time produced by the various extractsare. given in thetable.

times of normal plasma with diflercnz T he thromboplastin throm boplastins Thromboplastin time Thromboplastin species: of human plasma (seconds) Human brain thermoplastin, giving a thromboplastin time of 13-15 seconds, and rabbit brain thrombopiastin, producing a thromboplastin time of 12-13 seconds, cannot be used.

The bovine brain thromboplastin is most useful. A detailed technique of its preparation is given below. The optimal concentration of this thromboplastin giving maximal activity contains 1.3-1.7 g. of brain substance, weighed in the dried state, per 100 m1. of solution.

(2) Cephalin.All cephalin preparations may be used if they fulfill the following requirements: (a) The cephalin suspension must be stable for at least 24 hours at to 25 C. (b) The cephalin time produced with normal non-activated (prepared by silicon technique) platelet-poor [centrifuged for minutes at 3000 rpm. (1 ,800 g. at 4 C.)] human plasma must be shorter than seconds. [Technique as described from this laboratory by B. A. Waaler: Scand. J. of Clin. & Lab. Investigation 9, 322 (1957).] (c) The cephalin time of non-activated platelet-poor plasma from a patient congenitally deficient in proconvertin (factor Vii) is the same as for normal plasma, below 55 seconds. This test is applied to exclude that part of the activity produced on normal plasma Which is caused by contamination of the cephalin preparation with thromboplastic activity.

The following cephalin preparations have been tested and found suitable: (a) Crude cephalin prepared from human brain. (b) Cephalin prepared from soya beans.

' [One such preparaiton is available commercially: Asolectin (Associated Concentrates Inc, 5701 32nd Avenue, Woodside, New York).] (0) Phosphatidylethanolamine prepared by the method of OBrien. [OBrien, 3. R: J. Clin. Path. 9, 47 (1956).]

The coagulation active principles in the cephalin preparations are phosphatidylethanolarnine, phosphatidylserine, and possibly other similar substances. There are great difiicult-ies in obtaining large quantities of individual phospholipid-s. The two first preparations are completely satisfactory for this reagent, however. Detailed techniques for (the preparation of crude human brain cephalin is given below. The optimal concentration of crude cephalin in the final reagent, is 0001-001 g. per 100 ml. of reagent and of asolectin 0010-0015 g. of dried substance per 100 ml. of reagent.

(3) Adsorbed pZasma.-Any plasma with the following characteristics may be used:

(A) It must be completely free of the following 4 clotting factors: prothrornbin (factor 11), proconverten (factor V11), Stuart-ProWer-factor (factor X) and antihemophilic-Bfactor (factor IX), as tested by the following methods- (1) Thromboplastin time (Quicks method, see above): more than 24- hours.

(2) The Russell viper venom cephal-in time [method- I-ijort, R, Rapaport, S. 1., Owren, P. A.: The Journal of Laboratory 5: Clinical Medicine 46, 39 (1955)] 2 lvlore than 3 hours.

(3) An-tihemophil-ia-B-factor [method-Stapp, W. F: Scand. J. Clin. Lab. Invest. 10, 169 (1958)]: No measurable activity.

(B) it must contain a concentration (relative to human plasma) of the following activities of at least percent.

(1) Proaccelerin (factor V) activity [method0wren, P. A.: The Coagulation of Blood (1947)].

(2) Antihemophilia-A-faotor activity [method-Waal or, B. A; Scand. J. Clin. Lab. Investigation ii (1959)].

(C) it must not contain measurable amounts of activation product (as tested by the method of Waaler, B. A.: Scand. J. Clin. Lab. Investigation Suppl. 11, 1959, page 32).

Adsorbed plasma fulfilling the requirements given above have been prepared from bovine, horse, dog, sheep and human plasma. Bovine plasma is preferred because of higher concentrations and greater stability of proaccelerin and antihcmophilia-A-tactor on storage.

Technique of preparation is identical for all species. Details of preparation from bovine plasma are given below.

(4) Calcium chloride-A one molar eous stock solution was prepared by dissolving 111.1 g. of anhy drous CaCl (analytical grade) in 1000 of distilled water. v

" echnique of preparation is given by examples as follows:

'(1) Preparation of bovine brain thromboplastin [meth od similar to Owren, P. A; Scand. J. Lab. "Clin. Invest.

Bovine brain obtained shortly after death was stripped of the pia and blood vessels and rinsed in running tap Water. The cerebellum and the medulla were discarded. The brain was cut in small pieces and again rinsed in running tap wa er. To 250 g. of brain tissue were added 1000 ml. of saline (45 (3.), and the mixture was blended for 4 minutes in a mix master (Ate-Mix blender and disintegrator). After standing for 1 hour the mixtu-re was centrifuged for 30 minutes at 2500 rpm. and the supernatant collected. The precipitate was discarded. The pH of the supernatant was adjusted to 7.35 with 0.5 normal NaOl-l, and volume of veronal bufier (pH 7.35 and ionic strength 0.154) was added. This buffer was made by rnixing11.75 g. sodium diethyl barbiturate, 14.67 g. sodium chloride, 430 ml. 0.1 normal HCl and distilled Water to 2,000 ml. The activity of a sample of the thromboplastin solution was tested in serial dilutions in physiological saline by Quicks method (0.20 ml. oxalated normal human plasma, 0.20 ml. thromboplastin solution, 0.20 ml. calcium chloride solution, 20 mm.) The dilution 1:1 gave maximal activity of 34.5 seconds. The thromboplastin solution was consequently diluted with an equal volume of physiological saline solution. The final solution contained 1.4 g. of dried brain substance per ml. of suspension.

(2) Preparation of human brain cephalin [lI1$ii10(l-- Hjort, P., Rapaport, S. 1., and Owron, P. A.: J. Lab. Clin. Med. 46, 89 (1955)].

The crude cephalin suspension is the acetone-insoluble, ether-soluble fraction of human brain. It was prepared as follows: Four hundred grams of human brain were Washed free of blood and membranes and ground with 300 ml. of acetone. (Merck & Company, Inc.) Afiter centrifugation, the acetone Was discarded and the residue re-extracted with the same volume of acetone a total of six times. The residue was then extracted with 1,800 ml. of ether overnight at room temperature. The ether was siphoned oil and evaporated to dryness by suction at 35 C. The resulting residue was washed twice for 15 minutes with 900 m1. of acetone and then redissolved in 200 m1. of ether. The ether was again evaporated to dryness and the suction continued for 75 minutes. About 9.3 g. of a Waxy cream-colored t0 tained 2.8 g. percent of crude cephalin in a milky white suspension, was frozen in small quantities at -20 C.

Before use a sample of the melted suspension was tested in serial dilutions with physiological saline for determining the. optimal dilution giving maximal activity in the cephalin time test. The shortest cephalin time with normal human plasma, being 52 seconds, was effected by a dilution of 1:200. The cephalin time of proconvertin deficient plasma was 51 seconds. The cephalin suspension was diluted 1:200 in the thromboplastin suspension to a concentration of 0.014 g. percent.

The final reagent before freezedrying contained -7 of this thromboplastin suspension and consequently a concentration of 0.0056 g. percent cephalin.

(3) Preparation of adsorbed bovine plasma:

(a) Collection of blood. One hundred ml. of 2.5 percent (W./v.) potassium oxalate monohydrate solution are added into perfectly siliconized (Siloxan 300, Uddeholms Aktiebolag, Uddeholm, Sweden) one liter glass bottles. The bottles are kept in packed ice, and filled with 900 ml. of bovine blood from the jugular vein by r puncture with a coarse siliconized needle (Monocote, Armour Laboratories, Eastbourne, England). All the following steps are performed at low temperature (between +2 C. and 4 C.) and by the use of siliconized equipment.

(b) The bottles are centrifuged for 30 minutes at 2,5 00 rpm. (1,700 g.).

(c) The plasma is siphoned off into beakers.

(d) The plasma is adsorbed with analytical grade BaSQ, (Baker) in an amount of 75 mg. per ml. of plasma for minutes and by slow stirring with a glass rod.

(e) The plasma is centrifuged for 30 minutes at 2,500 rpm. (1,700 g.) and the sediment discarded.

(f) A second adsorption with BaSO is performed in the same Way.

(g) A sample of the adsorbed plasma is tested with the Russel viper venom cephalin method. If any visible clot appears within 2 hours, a third adsorption is performed.

(h) The adsorbed plasma is passed through a percent asbestos paper filter pad (Carlson Ltd., London), filter diameter 15 cm., CO pressure 1 kg. per square cm., filter changed every 500 ml.

(2') The plasma is dialyzed for 12 hours against ten times the plasma volume of 0.85 percent saline solution, the saline being changed three times (Cellulose Casings, Visiting Company, Chicago).

(j) The dialyzed plasma is adjusted to pH 7.35 with N/Z HCl (Radiometer, Copenhagen, Denmark). Accuracy :0.05.

(k) The plasma is tested with the test methods referred to above. If the plasma does not fulfill the requirements stated, it is discarded.

(4) The preparation of calcium chloride solution of 12.5 milli-molar:

A 50 millimolar solution of calcium chloride is prepared by first mixing 50 ml. of the calcium chloride stock solution of 1 molar and 950 ml. of distilled water. One volume of this solution and 3 volumes of physiological saline solution are then mixed (this technique provides correct ionic strength).

Preparation of the reagent:

Equal volumes of adsorbed bovine plasma and the thromboplastin cephalin suspension are mixed. To the mixture is added 6 its volume of a calcium chloride solution of 12.5 millimolar.

that may be used to prepare the freezedried product.

The freeze-drying of the mixture mentioned above is 10 performed in ampules of lenaglass of .30 ml. volume. The ampules are of the usual type, capable of beingsealed *so as to preserve its contents.

10 ml. of said mixture are poured carefully into the ampule. The ampule with its contents is first cooled .to a temperature of about C. During the cooling to said temperature, the ampule is situated in a slightly slanting position, approximately 4 to 6 degrees relative to the horizontal, and further the ampule is brought to rotate during said cooling. The cooling period is about /2 to 1 hour.

The freeze-drying is then carried out under high vacuum and with supplying of heat to the ampule, for example so that the ampule is in contact with suitable elements, to 6070 C., so long as ice is still present in the mixture; and then the temperature is lowered to about 40 C. during the final drying step. i

The product is treated under vacuum until the water content is below 0.5%. The ampule is then sealed, preferably by closing the ampule opening by means of fusing undervacuum.

Comments The preparation. of cephalin, thromboplastin and calcium solutions follows previously known principles. In the preparation ofadsorbed plasma the following points .are stressed: i

(1) Careful silicone technique has to be applied in order to prevent the activation of clotting factors by contact with glass. Such activation includes a reaction comprising the antiheinophilia-C-factor and the Hageman-factor producing an activation product which shortens the cephalin time. The ".activation product becomes slowly inactivated. A reagent containing activation productj. therefore, is unstable. The significance of this fact has not previously been understood.

(2) The adsorption procedure has been carefully standardized in order to obtain complete removal of the fourfactors mentioned. A reagent containing adsorbed plasma which does not clot on addition of thromboplastin and calcium. hasnot previously been described.

(3) Careful centrifuging and filtering for complete removal of the suspended barium sulphate is essential. Residual particles. of barium sulphate in the reagent with clotting factors adsorbed on the surface ofthe particle will produce thrombin 'formationon recalcification followed by the formation of fibrin.

(4) 'As a new principle in the. preparation of this reagent, dialysis is introduced in order to obtain? (a) Removal of the plasmas carbonic acid-bicarbonate buffer system. This is necessary for exact adjustment and stabilizationof pH at 7.35 =inthe finished dried preparation. If CO remains, it will escape during Ifreeze- .drying followed by rising pH.

(b) Removal of oxalate. This isnecessary for (l) stability of the proaccelerin, .(2) to prevent precipitation of calcium oxalate with consequentadsorption of coagulation factors during performance of the test, .and (3) to permit use of citrate plasma for the determination.

' (5) After dialysis the pH is adjusted .to pH 7.35. This has significance for thestability ofthe reagent and for :the solubility of fibrinogen on reconstruction.

-(6) The final alLin-one reagentis freeze-dried in ampules for-distribution.

(7) Stability :of the freeze-dried reagent. The reagent shows unchanged activity afterstorage of the ampules at 47 C. for 30 days.

(8) TheIfreeze-dried reagent is" reconstituted by addition :of distilled 'WEL'ECI or calciumchloride solution to the ampule for the capillary and venous blood method, respectively.

(9) Stability of the reagent in solution.

'The reagent in solution beforefreeze-drying as -Well as after reconstitution of the dried reagent shows unchanged reactivity even after storage at room temperature for 12 hours. This stabilityis obtained, inter alia, by the total amass? ill elimination which has been achieved of the above-mentioned four coagulation factors. It has formerly been possible to mix thromboplastin and calcium into one reagent, but a stable reagent has not been previously possible after recalcification of an adsorbed plasma with or without thromboplastin.

Procedure for use When using the preparation for determining the coagulability of the blood, the following procedure is employed:

After reconstitution of the reagent in distilled water or calcium chloride solution (see below), 0.5 ml. is placed by means of a pipette in a very small test tube (diameter 8-9 mm., length 60 mm.). A measured quantity of the plasma or blood to be examined (see below) is added and the coagulation time determined with a stop-watch. The recorded time is an expression of the effect of the anticoagulant treatment on the coagulation process. The test is performed in a water-bath at 37 C.

The registered coagulation time is converted to relative activity, stated in percentage, using a correlation curve. Examples of correlation curves are given in FIG- URE 3. These correlation curves show the ratio between recorded coagulation times in two systems and the coagulation activity expressed in percentage of the normal.

The concentration of normal plasma in percentage is plotted on the logarithmic scale of the abscissa and the coagulation times in seconds (30-300 sec.) are plotted on the logarithmic scale of the ordinate. Curve K shows the method used for capillary blood, 0.10 ml. volume. Curve H shows the method used for citrate blood, 0.10 ml. volume, and for citrate plasma in dilution: 3 parts of plasma to 2 parts of saline,'and 0.10 ml. volume.

Curve C shows an alternative method used for citrate plasma in dilution: 1 volume of plasma to 4 volumes of saline, and 0.20 ml. of the dilution as test volume.

The method can be used both for capillary blood (whole blood), which contains no added anticoagulant, and for citrate blood or citrate plasma. The reagent is provided with optimal concentration for capillary blood and cannot be used directly for citrate blood or citrate plasma, since for such samples the calcium concentration is inoptimal.

For citrate blood and citrate plasma, therefore, the dried reagent has to be reconstituted with 3.2 millimols calcium chloride solution which compensates for the amount of citrate in the test sample.

(1) Capillary blood:

A skin incision which affords lively bleeding should be made in the finger tip or the ear lobe. 0.10 ml. of Whole blood is then filled into a pipette in the usual way, and this is at once mixed with the 0.5 ml. reagent which has previously been placed in the water-bath at 37 C. The coagulation time is determined.

(2) Citrate blood:

Venous blood is used mixed with 3.13 percent (w./v.) sodium citrate dihydrate solution in the ratio 1 part sodium citrate solution to 9 parts of blood. Example: 0.2 ml. sodium citrate solution is drawn into a 2 ml. syringe, and the syringe is then filled with blood by venipuncture to a volume of 2 ml. The sample is transferred to a small test tube and 0.10 ml. citrate blood is removed with a pipette and mixed with 0.5 ml. reagent, and the coagulation time is determined in the manner described above.

(3) Citrate plasma:

Citrate blood is prepared in the manner described in paragraph (2). After centrifuging, 0.6 ml. citrate plasma is 'measured off into a small test tube and 0.4 ml. physiological salt solution added. After mixing, 0.10 ml. of the diluted plasma is removed by pipette and at once mixed 'with .05 ml. of the reagent. The coagulation time is determined as indicated above.

The new all-in-one reagent constitutes a new creation in that it makes it possible to perform a combined deteri2 'mination of the coagulation activity of both the internal and the external coagulation systems.

The preparation of the reagent is based on principles which have not previously been employed. The reagent has a number of advantages when compared with earlier reagents: (1) It is very stable at room temperature, thus simplifying distribution and storage. (2) It withstands heating up to 47 C. for a minimum of 30 days, and can therefore be used in tropical regions. (3) The requirement for only one eagent makes the procedure simpler and the performance of the test faster than was the case for earlier methods. (4) It is the only method so far devised which provides an adequate index of the'coagulation disturbance produced by oral anticoagulant agents. In contrast to earlier methods this one. is sensitive to reduction of antihemophilia-B-factor. (5) As a consequence of the above qualities, this method gives far greater protection against hemorrhagic complications than other methods. (6) The method is time-saving. (7) Because of the stability of the reagent in reconstituted form it affords reliable and reproducible results.

I claim: 7

l. A process which comprises (i) adrr'xing (a) thromboplastin selected from the group consisting of brain thromboplastin and lung thromboplastin from at least one of bovine, horse, swine, dog and sheep,

(b) free cephalin from human brain and (c) adsorbed plasma, plasma being selected from the group consisting of bovine, horse, swine, dog and pie '2, which plasma has a high concentration of ail factors which are not reduced during anticoagulant treatment, and

(ll) aiding to the resulting admixture calcium chloride in an amount to yield optimal concentration for coagulation after addition of test plasma.

2. A combination of thrombopiastin from an animal organ and free cephalin in a single composition, the thromboplastin being a member selected from the group consisting of brain throniboplastin and lug thromboplastin from at least one of bovine, swine, dog and sheep, the thromboplastin being stable on storage at from about to about C. for at least 24- hours and having a maximum activity on normal blood plasma to yield a thromboplastin time between and seconds, and the cephalin having a high reactivity toward the internal clotting system of human plasma to yield a cephalin time of less than seconds.

3. A composition for determining the coaguiability of blood for control of treatment with anticoagulant agents, the composition comprising as essential constituents the combination consisting essentially of ([1) animal organ thromboplastin which has a weak but constant reactivity toward the external clotting system of human blood plasma, the thromboplastin being a member selected from the group consisting of brain thromboplastin and lung thromboplastin from at least one of bovine, horse, swine, dog and sheep, and (5) free cephalin having a high reactivity toward the internal clotting system of human plasma, the correlated proportions of the thromboplastin and the free cephalin assuring approximately cc al influence by both internal and external coagulation systems on coagulation time.

4. A composition for determining the coagulability of blood for control of treatment with anticoagulant agents, the composition comprising:

(a) .thromboplastin selected from the group consisting of brain thromboplastin and lung thromboplastin from at least one of bovine, horse, swine, dog and sheep, the thromboplastin having a weak but constant reactivity toward the external clotting system of human blood plasma,

(b) adsorbed plasma, from which plasma prothrombin,

tuart-factor, proconvertin and antihemophilia- 13 B-factor have been removed, said plasma being a member selected from the group consisting of bovine, horse, swine, dog and sheep plasma, and

() free cephalin produced from human brain, said cephalin having a high reactivity toward the internal clotting system of human plasma,

the influence of both internal and external coagulation systems on coagulation time being maintained approximately equal by the relative concentrations of the thromboplastin and the free cephalin.

5. A blood coagulability-determining composition consisting essentially of (a) thromboplastin selected from the group consisting of brain thromboplastin and lung thromboplastin from at least one of bovine, horse, swine; dog and sheep, the thromboplastin having a weak but constant reactivity toward the external clotting system of human blood plasma,

(1')) adsorbed plasma, from which plasma prothrombin,

Stuart-factor, proconvertin and antihemophilia- B-factor have been removed, said plasma being a member selected from the group consisting of bovine, horse, swine, dog and sheep plasma, and

(c) a tree cephalin preparation having a high reactivity toward the internal clotting system of human plasma,

the influence of both internal and external coagulation systems on coagulation time being maintained approximately equal by the relative concentrations of the thromboplastin and the free cephalin.

6. A blood coagulability-determining composition comprising:

(a) thromboplastin selected from the group consisting of brain thromboplastinand lung thromboplastin from at least one of bovine, horse, swine, dog and sheep, the thromboplastin having a weak but constant reactivity toward the external clotting system of human blood plasma, and

(b) free cephalin from human brain and having a high reactivity toward the internal clotting system of human plasma,

the concentrations of said thromboplastin and said free cephalin being correlated in the composition to maintain approximately equal the influence on coagulation time of both internal and external clotting systems.

:7. A blood-coagulability-determining composition comprising:

(a) thromboplastin selected from the group consisting of brain thromboplastin and lung thromboplastin from at least one of bovine, horse, swine, dog and sheep, the thromboplastin having a weak but constant reactivity toward the external clotting system of human blood plasma, and

(b) a free cephalin preparation from soya beans and having a high reactivity toward the internal clotting system of human plasma,

the concentrations of said thromboplastin and said free cephalin being correlated in the composition to maintain approximately equal the influence on coagulation time of both internal and external clotting systems.

8. A blood coagulability-determining composition comprismg:

(a) thromboplastin selected from the group consisting of brain thromboplastin and lung thromboplastin from at least one of bovine, horse, swine, dog and sheep, the thromboplastin having a weak but constant reactivity toward the external clotting system of human blood plasma,

(1;) free cephalin having a high reactivity toward the internal clotting system of human plasma, and

(c) calcium chloride in an amount which gives optimal concentration for coagulation after reconstitution and addition of test plasma,

the concentration of said thromboplastin and said free cephalin being correlated in the composition to maini4 tain approximately equal the influence on coagulation time of both internal and external clotting systems.

9. A blood coagulability-determining composition comprising:

(a) thromboplastin selected from the group consisting of brain thromboplastin and lung'thromboplastin from at least one of bovine, horse, swine, dog and sheep,the thromboplastin having a weak but constant reactivity toward the external clotting system of human blood plasma,

(E2) free cephalin from human brain and having a high reactivity toward the internal clotting system of human plasma, and

(0) calcium chloride in an amount which gives optimal concentration for coagulation vaiter reconstitution and addition of test plasma,

the concentrations of said thromboplastin and said free cephalin being correlated in the composition to maintain approximately equal to the influence on coagulation time of both internal and external systems.

10. A blood coagulability-deternnning composition comprising:

(a) thromboplastin selected from the group consisting of brain thromboplastin and lung thromboplastin from at least one of bovine, horse, swine, dog and sheep, the thromboplastin having a weak but constant reactivity toward the external clotting system of human blood plasma,

(b) a free cephalin preparation from soya beans and having a high reactivity toward the internal clotting system of human plasma, and

(c) calcium chloride in an amount which gives opimal concentration for coagulation after reconstitution and addition of test plasma,

the concentrations of said thromboplastin and said free cephalin being correlated in the composition to maintain approximately equal the influence on coagulation time of both internal and external clotting systems.

11. A blood coagulability-determining composition comprising:

(a) thromboplastin selected from the group consisting of brain thromboplastin and lung thromboplastin from at least one of bovine, horse, swine, dog and sheep, the thromboplastin having a weak but constant reactivity toward the external clotting system of human blood plasma,

(b) adsorbed plasma, from which plasma prothrombin, Stuart-factor, proconvertin and antihemophilia-B- factor have been removed, said plasma being a member selected from the group consisting of bovine, horse, swine, dog and sheep plasma,

(0) free cephalin from human brain, and

(d) calcium chloride in an amount which gives optimal concentration for coagulation after reconstitution and addition of test plasma,

the concentrations of said thromboplastin and said free cephalin being correlated in the composition to maintain approximately equal the influence on coagulation time of both internal and external clotting systems.

12. A blood coagulability-determining composition comprising:

(a) thromboplastin selected from the group consisting of brain thromboplastin and lung thromboplastin from at least one of bovine, horse, swine, dog and sheep, the thromboplastin having a weak but constant reactivity toward the external clotting system of human blood plasma,

(5) adsorbed plasma, from which plasma prothrombin, Stuart-factor, proconvertin and antihemophilia- B-factor have been removed, said plasma being a member selected from the group consisting of bovine, horse, swine, dog and sheep plasma,

(c) a free cephalin preparation from soya beans and having a high reactivity toward the internal clotting system of human plasma, and

15 '(a') calcium chloride in an amount Which gives optimal concentration for coagulation after reconstitution and addition of test plasma, the concentrations of said thromboplastin and said free cephalin being correlated in the composition to maintain approximately equal the influence on coagulation time of both internal and external clotting systems.

13. A blood coagulabilityeterrnining composition comprisin (a) thromboplastin selected from the group consisting of brain thromboplastin and lung thromboplastin from at least one of bovine, horse, swine, dog and sheep, the thromboplastin having a weak but constant reactivity toward the external clotting system of human blood plasma, a (b) adsorbed plasma free from prothrornbin, Etuartfactor, proconvertin and antihernophilia-B-factor and containing a high concentration of all coagulation factors which are not affected by peroral anticoagulant agents, said plasma being selected from the group consisting of bovine, horse, swine, dog and sheep plasma, (c) free cephalin having a high reactivity toward the internal clotting system of human plasma, and (a') calcium chloride in an amount which gives optimal concentration for coagulation after reconstitution and addition of test plasma, the concentrations of said thromboplastin said free cephalin being correlated in the composition to maintain approximately equal the influence on coagulation time of both internal and external clotting systems.

14. A blood coagulability-determining composition comprising:

(a) bovine throniboplasi having a Weak but constant reactivity toward the external clottin system of human blood plasma and (12) free cephalin having a high reactivity toward the internal clotting system of human plasma.

Relierences Cit d in the file of this patent UNITED STATES PATENTS 37,980 Blanchard Aug. 31, 1954 47,350 Singher Aug. 12, 1958 OTHER REFERENCES Waaler: J. Clin and Lab. lnvst, vol. 9, 1957, pp. 322330.

Tocantins: The Coagulation of Blood, Grund and Stratton, New York, 1955, pp. 1789, 183484.

Gong ea: 1. Lab. 8: Clin Med, 48:4, October 1956, pp. 62446.

Merck index, Merck and Co., Railway, N.J., 6th ed, 1952, pp. 9556.

Langdell: J. Lab. & Clin, Med, 41:4, April 1953, pp. 637-647.

Stormorken: J. Lab. & Clin, Med, 48:4, October 1956, pp. 5l9528.

Ware: The Amer. J. of Clinical Path, 22:8, August 7 Sise: 1. Lab. & Clin. Med, 49:1, January 1957, pp. 6983.

Seegers: Amer. J. Physio, vol. 178, July 1954-, pp. 1-9.

Caldwell: Reprint Amer. Heart 1., 56:5, November 1958, pp. 737-741.

Newlands: Nature, vol. 176, 1955, p. 885. 

1. A PROCESS WHICH COMPRISES (I) ADMIXING (A) THROMBOPLASTIN SELECTED FROM THE GROUP CONSISTING OF BRAIN THROMBOPLASTIN AND LUNG THROMBOPLASTIN FROM AT LEAST ONE OF BOVINE, HORSE, SWINE, DOG AND SHEEP, (B) FREE CEPHALIN FROM HUMAN BRAIN AND (C) ADSORBED PLASMA, SAID PLASMA BEING SELECTED FROM THE GROUP CONSISTING OF BOVINE, HORSE, SWINE, DOG AND SHEEP PLASMA, WHICH PLASMA HAS A HIGH CONCENTRATION OF ALL FACTORS WHICH ARE NOT REDUCED DURING ANTICOAGULANT TREATMENT, AND (II) ALDING TO THE RESULTING ADMIXTURE CALCIUM CHLORIDE IN AN AMOUNT TO YIELD OPTIMAL CONCENTRATION FOR COAGULATION AFTER ADDITION OF TEST PLASMA.
 2. A COMBINATION OF THROMBOPLASTIN FROM AN ANIMAL ORGAN AND FREE CEPHALIN IN A SINGLE COMPOSITION, THE THROMBOPLASTIN BEING A MEMBER SELECTED FROM THE GROUP CONSISTING OF BRAIN THROMBOPLASTIN AND LUG THROMBOPLASTIN FROM AT LEAST ONE OF BOVINE, SWINE, DOG AND SHEEP, THE THROMBOPLASTIN BEING STABLE ON STORAGE AT FROM ABOUT 20* TO ABOUT 25*C. FOR AT LEAST 24 HOURS AND HAVING A MAXIMUM ACTIVITY ON NORMAL BLOOD PLASMA TO YIELD A THROMBOPLASTIN TIME BETWEEN 30 AND 50 SECONDS, AND THE CEPHALIN HAVING A HIGH REACTIVITY TOWARD THE INTERNAL CLOTTING SYSTEM OF HUMAN PLASMA TO YIELD A CEPHALIN TIME OF LESS THAN 55 SECONDS. 